JP2007527722A - New expression tools for multiprotein applications - Google Patents

Abstract

  The present invention relates to polynucleotides, vectors, host cells, and recombinant animals containing said polynucleotides for multiple gene applications, including novel functional arrangements. In addition, the present invention is directed to methods for producing multigene expression cassettes, methods for producing multiprotein complexes in vitro and in vivo, and methods for producing vaccines. Furthermore, the present invention provides a method for screening protein complex interactions or protein modifications, and can interact with a protein complex, or can modify a protein, or inhibit a protein complex interaction, or a protein In vitro or in vivo screening methods for candidate compounds capable of inhibiting the modification of the above. The invention also provides (i) to produce a drug for gene therapy, (ii) to produce a recombinant of a multiprotein complex, (iii) to produce a vaccine, or (iv) to be of interest It relates to the use of the polynucleotides, vectors, host cells or recombinant animals of the invention for screening compounds. Last but not least, the present invention is directed to a kit comprising at least a component comprising a polynucleotide, vector, and / or host cell of the present invention.

Description

  The present invention relates to polynucleotides for multiple gene applications, including novel functional arrangements, as well as vectors, host cells, and recombinant animals comprising said polynucleotides.

  In addition, the present invention is directed to a method for producing a multi-gene expression cassette, a method for producing a multiprotein complex in vitro and in vivo, and a method for producing a vaccine.

  Furthermore, the present invention provides a method for screening protein complex interactions or protein modifications, and can interact with a protein complex, or can modify a protein, or inhibit a protein complex interaction, or a protein In vitro or in vivo screening methods for candidate compounds capable of inhibiting the modification of the above.

  The invention also provides (i) to produce a drug for gene therapy, (ii) to produce a recombinant of a multiprotein complex, (iii) to produce a vaccine, or (iv) to be of interest It relates to the use of the polynucleotides, vectors, host cells or recombinant animals of the invention for screening compounds.

  Last but not least, the present invention is directed to a kit comprising at least a component comprising a polynucleotide, vector, and / or host cell of the present invention.

  The identification of novel multiprotein complexes in eukaryotic cells is rapidly advancing, particularly with the advent of genome-wide analysis of protein-protein interactions, the introduction of powerful multiple affinity protein purification methods, and This is due to the development of high-sensitivity analysis technology. Based on extensive two-hybrid studies, the average number of partners interacting with a particular protein was estimated to be 5-8 in baker's yeast (Schwikowski et al. A network of protein-protein interactions in yeast). Nat. Biotechnol. 18, 1257-1261 (2000); Bader et al. Analyzing protein-protein interaction data obtained from different sources. Nat. Biotechnol. 20, 991-997 (2002); Von Mering et al. Comparative assessment of large-scale data sets of protein-protein interactions.Nature 417, 399-403 (2002); Grigoriev, A. On the number of protein-protein interactions in the yeast proteome.Nucleic Acids Res. 31, 4157-4161 (2003) ). This has led to the concept of cells as a collection of protein components consisting of multiple components, essentially one for the main process. This poses an important challenge for protein production technologies aimed at the structural and functional studies of eukaryotic multiprotein complexes at the molecular level, because the amount of intracellular This is because it is generally unsuitable for large-scale extraction from sources.

  Polycystin vectors carrying multiple genes encoding multiprotein complex components have been used in selective cases of transport in gene therapy (De Felipe, P. Polycistronic viral vectors. Curr. Gene Ther. 2 , 355-378 (2002); Planelles, V. Hybrid lentivirus vectors. Methods Mol. Biol. 229, 273-284 (2003)). Recently, polycistronic vectors have been used to express a transcription factor complex consisting of four subunits in E. coli (Selleck et al. A histone fold TAF octamer within the yeast TFIID transcriptional coactivator. Nat. Struct. Biol. 8, 695-700 (2001)). The DNA constructs in these studies were made by routine methods using endonucleases and ligases. However, protein complexes in eukaryotes often contain ten or more subunits with individual polypeptides up to several hundred kDa in size, greatly limiting the applicability of conventional cloning strategies, And most of E. coli are excluded as useful host systems for heterologous protein expression.

Recombinant baculoviruses are particularly attractive for high-level production of large protein assemblies (O'Reilly et al. Baculovirus expression vectors. A laboratory manual. Oxford Press, New York (1994)). Genes driven by the Autographa californica nuclear polyhedrosis virus (AcNVP) late promoter are often abundantly expressed, reliably processed, and directed to their appropriate intracellular compartment. Even structurally complex particles such as capsid structures are well assembled in insect cells using the baculovirus system (Roy et al. Baculovirus multigene expression vectors and their use for understanding the assembly process of architecturally complex virus particles. Gene 190, 119-129 (1997)). Multiple gene expression in one cell can be achieved by co-infecting each virus carrying one foreign gene. However, this attempt necessarily results in considerable variability at individual protein production levels as a result of statistical dispersion in virus stoichiometry between infected cells. An excellent alternative is to infect a single baculovirus containing all the selected heterologous genes (Roy et al., See above; Bertolotti-Ciarlet et al. Immunogenicity and protective efficacy of rotavirus 2 / 6-virus −like particles produced by a dual baculovirus expression vector and administered intramuscularly, intranasally, or orally to mice. Vaccine 21, 3885-3900 (2003)). This is made possible by the flexible nature of the AcNVP envelope, which can adapt large DNA inserts to a circular 130 kb dsDNA baculovirus genome. So far, recombinant baculovirus production has been carried out in two steps. This process involves first cloning a foreign gene into a small transfer vector grown in E. coli and then inserting it into a large baculovirus genome by homologous recombination in insect cells. Gives 30-80% recombinant progeny when using linearized parental viral DNA (O'Reilly et al., See above). This procedure was actually simplified by the introduction of a baculovirus shuttle vector (bacmid bMON14272) grown in E. coli (Luckow et al. Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in E. coli. J. Virol. 67, 4566-4579 (1993)). The bacmid contains a Tn7 adhesion site for transferring foreign genes from a transfer vector (Bac-to-Bac®, Invitrogen) (Luckow et al., See above; Bac-to Bac ^™ Baculovirus Expression Systems Manual, Invitrogen, Life technologies Incorporated (2000)). Bicistronic, containing polyhedrin (polh) and p10 late viral promoter in two separate expression cassettes (with restriction site set for sequential subcloning of two foreign genes for co-expression) The transfer vector pFastBac ^™ Dual was introduced.
O'Reilly et al. Baculovirus expression vectors. A laboratory manual. Oxford Press, New York (1994) Luckow et al. Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in E. coli.J. Virol. 67, 4566-4579 (1993)

  In a discontinuous modular fashion that alleviates the limitations caused by the lack of useful restriction sites in a group of coding sequences each containing thousands of base pairs to produce large, multi-protein complexes containing many subunits There is still a great need for transfer vectors that can assemble genes.

  Accordingly, it is an object of the present invention to provide new tools and methods for the rapid and flexible production of multiprotein complexes.

  There is also a need for new tools and methods for producing multiprotein complexes of 10 or more subunits with individual polypeptides up to several hundred kDa in size by recombinant techniques.

  It is a further object of the present invention to provide new tools and methods for producing large multiprotein complexes with multiple subunits and simultaneously minimizing the unique restriction sites required.

  A further object of the present invention is to provide a flexible system for producing multiprotein complexes that can replace the coding sequence in a modular manner that avoids a new reconstruction of the entire assembly of coding sequences for individual subunits. Is to provide.

  In addition, specific organisms in various organisms, eukaryotes (mammalian, yeast, etc.), and prokaryotes, or under selected conditions (eg, early, late, late, etc. of viral infection of host cells) There is also a need for tools and methods that allow for easy module exchange of biospecific promoters, tissue specific promoters, or cell type specific promoters to produce multiprotein complexes by recombinant techniques.

  The solubility and activity of biopolymers are often reduced when the interacting components are separated, manufactured and isolated and purified, so that they are physiologically meaningful even when reconstituted under in vitro conditions. Some data cannot be obtained. Therefore, for studying interactions between macromolecules, it is ideally capable of co-expression of two or more protein assemblies in a single cell, each consisting of multiple components, and is powerful and scalable. A certain system is needed.

[Summary of the Invention]
As a first aspect, the above-described object is achieved by the following formula (I):

［この配置は以下を含む：
（a）頭部−頭部、頭部−尾部、又は尾部−尾部の配置での少なくとも２つの発現カセット、T1−MCS1−P1及びP2−MCS2−T2；それぞれ、マルチクローニングサイトであるMCS1又はMCS2を含み、MCS1はプロモーターP1及び終了配列T1に隣接しており、MCS2はプロモーターP2及び終了配列T2に隣接している、
（b）プロモーターP1とP2との間にある少なくとも１つの増殖モジュールM（少なくとも２つの制限部位A及びBを含む）、
（c）少なくとも２つの制限部位X及びY（それぞれ発現カセットの１つに隣接している）、
{式中：
（i）制限部位A及びX並びにB及びYは適合する、しかし、
（ii）AY及びBXのライゲーション産物は、制限部位A、B、X及びYに特異的な制限酵素であるa、b、x、又はyによって酵素的に切断されず、かつ
（iii）制限部位A及びB並びにX及びYは適合しない}］
の機能的配置を含む、複数遺伝子適用のための新規なポリヌクレオチドを提供することにより解決される。

[This arrangement includes:
(A) at least two expression cassettes in head-to-head, head-to-tail, or tail-to-tail arrangement, T1-MCS1-P1 and P2-MCS2-T2; MCS1 or MCS2, which are multiple cloning sites, respectively MCS1 is adjacent to promoter P1 and termination sequence T1, and MCS2 is adjacent to promoter P2 and termination sequence T2.
(B) at least one growth module M (including at least two restriction sites A and B) between promoters P1 and P2,
(C) at least two restriction sites X and Y (each adjacent to one of the expression cassettes),
{Where:
(I) Restriction sites A and X and B and Y are compatible, but
(Ii) the ligation product of AY and BX is not enzymatically cleaved by a, b, x, or y, which are restriction enzymes specific for restriction sites A, B, X and Y, and (iii) restriction sites A and B and X and Y are not compatible}]
It is solved by providing novel polynucleotides for multiple gene applications, including the functional arrangement of

  The polynucleotide of the present invention may be DNA, RNA, or a polynucleotide comprising one or more synthetic nucleotide analogs, preferably DNA, more preferably double-stranded DNA.

  As used herein, the term “expression cassette” refers to a DNA fragment, which includes a promoter sequence that is used to supplement the DNA transcription machinery, followed by the RNA translation machinery. For insertion of a DNA fragment encoding a selected gene product, including an oligonucleotide encoding a signal sequence (eg, the Kozak consensus sequence in eukaryotes or the Shide-Dalgarno sequence in prokaryotes) At least one DNA sequence (multicloning site MCS) that is cleaved by the restriction enzyme used, and termination directed to processes related to termination of transcription and modification of RNA transcripts (eg, polyadenylation in eukaryotes) It should be understood that it comprises an oligonucleotide sequence comprising is there.

  As used herein, the term “propagation module” relates to a set of DNA fragments that are placed outside and / or between expression cassettes to allow repeated combinations of multiple or multiple expression cassettes. Should be understood.

  A compatible restriction site is a restriction site that, when cleaved by the same type of enzyme, results in an overhanging single-stranded region or a recessed single-stranded region of DNA of the same nucleotide length, ie, a complementary Watson-Crick Base pairing can be used to anneal to each other and thus recombine with a ligase to produce a covalently linked functional DNA molecule. Alternatively, restriction sites that result in blunt ended DNA fragments upon cleavage are also compatible, ie, they can be recombined by ligase to produce a covalently linked functional DNA molecule.

  Non-limiting examples of restriction endonucleases that yield compatible sticky ends are: SpeI / AvrII; AgeI / XmaI / SgrA; BamHI / BglII; BsrGI / BanI; EagI / NotI; EcoRI / MfeI; NdeI / AseI; NheI / XbaI; PstI / NsiI; SalI / XhoI.

  Non-limiting examples of restriction endonucleases that result in compatible blunt ends (eg, all combinations can bind to each other) are BstZ17I, EcoRV, FspI, HpaI, MluNI, PmeI, ScaI, SnaBI, StuI, XmnI.

  The terms “head-head”, “head-tail”, and “tail-tail” refer to the arrangement of promoters and terminators of separate expression cassettes facing each other. For example, T1-T2 is a “tail-tail” arrangement, P1-P2 is a “head-head” arrangement, and P1 / P2-T2 / T1 is a “head-tail” arrangement. Formula (I) shows a head-to-head arrangement, but of course a different arrangement (provided that the growth site M is between the expression cassettes and the restriction sites X and Y are each of the arrangement of formula (I). It is understood that (adjacent to the outer boundary of the expression cassette) is also within the scope of the present invention.

  Preferably, the at least two expression cassettes are in a “head-head” arrangement.

  In a preferred embodiment, the invention provides that the restriction sites A and B of the growth module M are selected from the group consisting of BstZ17I, SpeI, ClaI and NruI, or a restriction enzyme having the same cleavage site as the enzyme (“isoschizomer”) To a polynucleotide.

  In a further preferred embodiment, the present invention relates to a polynucleotide wherein the restriction sites X and Y are selected from the group consisting of PmeI and AvrII, or a restriction enzyme having the same restriction site as the enzyme (“isoschizomer”).

In another preferred embodiment, the present invention is a promoter P1 and P2, polh, p10 and p _XIV very late baculoviral promoters, vp39 baculoviral late promoter, Vp39polh baculovirus late / very late hybrid promoter, P _{cap / polh,} pcna, etl, p35, egt, da26 baculoviral early promoters; CMV, SV40, UbC, EF -1α, RSVLTR, MT, is selected from the group consisting of P _DS47, Ac5, P _GAL and P _ADH, relates to polynucleotides.

  Furthermore, a preferred embodiment of the invention relates to a polynucleotide wherein the termination sequences T1 and T2 are selected from SV40, HSVtk, or BGH (bovine growth hormone).

  In addition to the functional arrangement of formula (I), preferably the polynucleotide of the invention further comprises at least one site for integration into a vector or host cell. Such integration sites will allow convenient genomic or transient integration of the polynucleotide into vectors and host cells. More preferred is an integration site for genomic uptake.

  The integration site is adenovirus, adeno-associated virus (AAV), autonomous parvovirus, herpes simplex virus (HSV), retrovirus, radionovirus, Epstein Bar virus, lentivirus, Semliki Forest virus And polynucleotides that are compatible with incorporation of the polynucleotide into a virus selected from the group consisting of and baculoviruses are particularly preferred.

  More preferred are polynucleotides of the invention in which the integration site is compatible with the incorporation of the polynucleotide into baculovirus.

  Baculovirus expression systems are widely used for the expression of recombinant proteins in insect cells. Recently, recombinant baculovirus vectors modified to contain mammalian cell active promoter elements have been successfully used for transient and stable gene delivery in a wide range of primary and established mammalian cell lines. . The application of modified baculoviruses for in vivo gene delivery has also been demonstrated. In contrast to other commonly used viral vectors, baculoviruses are unable to initiate the replication cycle and produce infectious viruses in mammalian cells, whereas the unique properties of replication in insect cells. have. This virus is easy to manipulate, allows large insertions of foreign DNA, has little or no cytopathic effect in mammalian cells that can be observed with a microscope, and has good biosafety properties. (Kost et al. Recombinant baculoviruses as mammalian cell gene-delivery vectors. Trends in Biotechnology, 20 (4), April 2002). These properties make baculoviruses particularly useful for the practice of the present invention.

  In a more preferred embodiment, said integration site (s), which may optionally be included in the polynucleotide of the invention, is a transposon element of Tn7, a λ-integrase specific adhesion site and an SSR (site specific Recombinases), preferably selected from the group consisting of cre-lox specific recombination (LoxP) sites or FLP recombinase specific recombination (FRT) sites.

  Preferably, the integration site (s) are mammalian, preferably human cells; pigs, preferably CPK, FS-13, PK-15 cells; cows, preferably MDB, BT cells; sheep, preferably FLL- YFT cells; C. elegans; yeast, preferably S. cerevisiae, S. pombe, C. albicans, P. Pastoris cells; and polynucleotides into eukaryotic host cells selected from the group consisting of insect cells Suitable for incorporation.

  For human host cells, the integration sites are HeLa, Huh7, HEK293, HepG2, KATO-III, IMR32, MT-2, pancreatic β cells, keratinocytes, bone marrow fibroblasts, CHP212, primary neurons, W12, SK-N -Selected from the group consisting of MC, Saos-2, WI38, primary hepatocytes, FLC4, 143TK-, DLD-1, fetal lung fibroblasts, primary foreskin fibroblasts, Saos-2 osteosarcoma, MRC5, and MG63 cells It is preferred to be compatible with the integration of the polynucleotide into the host cell to be treated.

  For insect host cells, the integration site is a host selected from the group consisting of S. frugiperda cells, more preferably Sf9, Sf21, Express Sf +, High Five H5 cells, and D. melanogaster cells, most preferably S2 Schneider cells. It is preferably compatible with the uptake of the polynucleotide into the cell.

  In a preferred embodiment, the polynucleotide of the present invention further comprises one or more resistance markers for selecting host cells having desired properties based on resistance to toxic substances. More preferably, the resistance marker is selected from ampicillin, chloramphenicol, gentamicin, spectinomycin, and kanamycin resistance marker.

  Where a prokaryotic host cell is desired for incorporation of the nucleotide of the present invention, the polynucleotide of the present invention provides an R6Kγ conditional replication origin for causing pir gene-dependent growth in a prokaryotic host. It is preferable to further include.

In a highly preferred embodiment, the polynucleotide of the invention comprises:
(A) promoters P1 and P2 selected from polh and p10,
(B) a termination sequence selected from SV40 and HSVtk,
(C) restriction sites A and B in the growth module M, selected from the group consisting of BstZ17I, SpeI, ClaI and NruI,
(D) restriction sites X and Y selected from the group consisting of PmeI and AvrII,
(E) Site for viral integration selected from cre-lox and Tn7 recombination systems.

  In a further aspect, the present invention is directed to a vector comprising the polynucleotide sequence of the present invention.

  In a preferred embodiment, the vector is selected from the group consisting of a plasmid, an expression vector and a transfer vector.

  These vectors are useful for integration of heterologous DNA elements, preferably genes. The vector may be an expression vector or a transfer vector, and is preferably one useful for eukaryotic gene transfer, transient or viral vector-mediated gene transfer.

  More preferred said vector is a plasmid or a virus.

  In a particularly preferred embodiment, the virus is adenovirus, adeno-associated virus (AAV), autonomous parvovirus, herpes simplex virus (HSV), radionovirus, Epstein Bar virus, lentivirus, sem Selected from the group consisting of Liki Forest virus and Baculovirus.

  In the most preferred embodiment, the vector of the present invention is a baculovirus expression vector.

  Modification of baculovirus expression vectors surprisingly found that disruption of the function of two baculovirus genes, v-cath and chiA, results in increased retention of intracellular compartments during infection and protein expression It was done. The properties of proteins produced by such novel baculovirus systems have been significantly improved through the reduction of virus-dependent proteolytic activity and cell lysis.

  Thus, in a different aspect, the present invention is also directed to common baculoviruses in which the two baculovirus genes, v-cath and chiA, are functionally disrupted.

  In a preferred embodiment, the present invention is directed to a baculovirus vector of the present invention in which two baculovirus genes, v-cath and chiA, are functionally disrupted.

  Preferably, the vector of the present invention further comprises a site for SSR (site-specific recombinase), preferably a LoxP site for cre-lox site-specific recombination.

  More preferably, the cre-lox site is located in one or both of the two baculovirus genes, v-cath and chiA, disrupting its function.

  In another preferred embodiment, the present invention relates to a vector comprising a transposon element, preferably a Tn7 adhesion site.

  Preferably, the adhesion site is located within the marker gene. By doing so, it is possible to evaluate whether the integration by metastasis is successful through the marker gene.

  More preferably, the marker gene is selected from the group consisting of luciferase, β-GAL, CAT, fluorescent protein coding gene, preferably GFP, BFP, YFP, CFP, and variants thereof, and the lacZα gene. Variants of marker genes have less than 30%, preferably less than 20%, more preferably less than 10% sequence differences, but retain functional marker properties that are substantially the same as the original marker gene. Yes.

  In the most preferred embodiment, the vector of the invention has the sequence of SEQ ID NO: 1 (pFBDM). See also FIG.

  In a further most preferred embodiment, the vector of the invention has the sequence SEQ ID NO: 2 (pUCDM). See also FIG.

  In the following, the vectors of the invention are illustrated with reference to the most preferred baculovirus vectors pFBDM and pUCDM. However, the following description of these particular vectors of the present invention should not be construed to limit the scope of the present invention.

  The preferred novel baculovirus transfer vectors described above were constructed specifically for multigene applications. These include modified recipient baculovirus DNA that has been modified to enhance protein production, and two access to the baculovirus DNA in E. coli cells made for this purpose. A simple and quick method for integrating genes via sites (attTn7 and LoxP) is possible.

  The vector of the present invention makes it possible to elucidate a protein interaction network (interactome). Since many of the identified multiprotein complexes are not present in natural cells in quantities sufficient for detailed molecular biology analysis, these studies are intended for large-scale heterologous protein production. Rely on recombination technology. Currently, recombinant expression methods require undue investment in both labor and material before expressing the multiprotein, and after expression, to quickly change the multiprotein components to review expression studies. Does not provide flexibility.

  The vectors of the present invention, particularly the baculovirus expression systems of the present invention, greatly facilitate and facilitate multiprotein expression.

  The transfer vectors of the present invention, particularly pFBDM and pUCDM, include a growth module that greatly facilitates the modular binding of heterologous genes while minimizing the required unique restriction sites. Viral promoters (eg, p10 and polh late promoters) in these vectors can be exchanged for other promoter sequences (early, late) if necessary. Similarly, the termination sequence (currently SV40, HSVtk) can be replaced.

  Use of baculovirus vectors with functionally disrupted v-cath and chiA genes can increase the retention of intracellular compartments during infection and protein production. The properties of proteins produced by such vector systems have been significantly improved through the reduction of virus-dependent proteolytic activity and reduced cell lysis.

  In addition, the vectors of the present invention allow a completely new protocol for the rapid generation of recombinant DNA (preferably baculovirus DNA) by accessing the vector genome through two specific sites. To do.

  In addition to the Tn7 transposon site, the LoxP sequence was introduced into another site on the baculovirus genome that is capable of accepting multiple gene expression cassettes derived from pUCDM plasmids by site-specific recombination. Protocols have been developed to fully perform Tn7 transfer (from pFBDM derivatives carrying multiple gene cassettes) and LoxP site-specific recombination (from pUCDM derivatives carrying multiple gene cassettes) in a single step in E. coli. These protocols not only incorporate multiple gene cassettes with coding sequences for multiprotein complex subunits into vectors (eg, baculovirus vectors), but also specific enzymes (kinases) to modify the protein under study. Acetylase, etc.) can be used. See FIG. 1 for further illustration.

  The transfer vector pFBDM contains two expression cassettes in a head-to-head arrangement, which contain multiple cloning sites flanking the polh or p10 promoter and the SV40 or HSVtk polyA signal sequence, respectively. MCS1 and MCS2 are included. Growth module M is located between the promoter sequences. The sequences used for Tn7 transfer (Tn7L and Tn7R) include an expression cassette and a gentamicin resistance marker (see Figure 2 for more details).

  The transfer vector pUCDM has the same expression cassette containing the same growth module as pFBDM. This expression cassette is adjacent to the LoxP inverted repeat. The vector pUCDM contains a chloramphenicol resistance marker and an R6Kγ conditional origin of replication that causes pir gene expression dependent growth in prokaryotic hosts. See Figure 3 for more details.

  The vectors pFBDM and pUCDM are particularly suitable for making multigene expression cassettes with a growth module inserted between two promoters. The growth logic is illustrated in FIG. The only prerequisite for assembling multiple gene expression cassettes is that the restriction enzymes used for propagation (eg, PmeI, AvrII, SpeI, and either BstZ17I or NruI) are unique, Can be easily accomplished, for example, by performing site-directed mutagenesis prior to assembling the multigene cassette. The gene is cloned into MCS1 and MCS2 of pFBDM. The entire expression cassette is then excised by PmeI and AvrII digestion. The resulting fragments are placed in a growth module of pFBDM derivatives containing an additional set of genes via either SpeI / BstZ17I or SpeI / NruI sites. SpeI produces sticky ends that are compatible with AvrII, while BstZ17I, NruI and PmeI are blunt cutters. In the process, the restriction sites involved are removed and growth can be repeated by repeatedly using the modules present in the inserted cassette. Similar logic also applies to the generation of pUCDM derivatives with multiple gene expression cassettes. In addition, the promoter and termination sequences can be readily altered using appropriate restriction sites in these vectors, if desired. See FIG. 4 for more details.

  In addition, the baculovirus genome was modified to obtain improved protein production properties. Disrupting the two baculovirus genes, v-cath and chiA, resulted in increased retention of intracellular compartments during infection and protein production. The v-cath gene encodes the viral protease V-CATH, which activates cell death through a process dependent on chiA (encoding chitinase), a nearby gene on viral DNA. Both genes were disrupted so that chitin-affinity chromatography for purification could be used selectively to eliminate V-CATH activity and not interfere with the chiA gene product. The properties of the protein produced by MultiBac baculovirus are markedly improved through a reduction in virus-dependent proteolytic activity and a reduction in cell lysis. Instead of the disrupted viral DNA sequence, a LoxP sequence for cre-lox site-specific recombination was included. See FIG. 5 for more details.

As used herein, the term “MultiBac” synonymously represents an exemplary and preferred embodiment of the baculovirus viral vector, ie, a modified baculovirus genome lacking the v-cath and chiA genes. Furthermore, “MultiBac” related to “MultiBac system” is a factor required for performing the modified baculovirus viral vector and the recombination reaction (Tn7 transfer, site-specific recombination) described herein. DH10α-derived E. coli cell type (DH10MultiBac ^ET , DH10MultiBac ^Cre, etc.) containing the baculovirus vector, and the E. coli cell type comprising pFBDM and pUCDM, which are additionally exemplary and preferred vectors of the present invention. Is included.

  All vectors of the present invention, particularly the preferred transfer vectors pFBDM and pUCDM, are useful for introducing multiprotein complexes into host cells.

  In another aspect, the invention relates to a host cell comprising a polynucleotide sequence and / or vector of the invention.

  Preferred host cells for practicing the present invention are mammalian cells, preferably human cells, rodent cells, pig cells, bovine cells, sheep cells; C. elegans cells; yeast cells; insect cells; and E selected from the group consisting of. coli cells.

  Preferred yeast cells for practicing the present invention are S. cervisiae, S. pombe, C. albicans and P. pastoris.

  Preferred E. coli cells for practicing the present invention are Top10, DH5α, DH10α, HB101, TG1, BW23473, and BW23474 cells.

  Preferred insect cells for practicing the present invention are S. frugiperda cells, preferably Sf9, Sf21, Express Sf +, or High Five H5 cells, and D. melanogaster, preferably S2 Schneider cells.

  Preferred human cells for practicing the present invention are HeLa, Huh7, HEK293, HepG2, KATO-III, IMR32, MT-2, pancreatic β cells, keratinocytes, bone marrow fibroblasts, CHP212, primary neurons, W12, SK -N-MC, Saos-2, WI38, primary hepatocytes, FLC4, 143TK-, DLD-1, fetal lung fibroblasts, primary foreskin fibroblasts, Saos-2 osteosarcoma, MRC5, and MG63 cells Selected from.

  Of course, the cells containing the polynucleotides and / or vectors of the present invention are in no way limited to isolated cells or cellular tissues.

  In another aspect, the invention also relates to a recombinant animal comprising a polynucleotide sequence and / or vector of the invention.

  Such recombinant animals are useful for elucidating the role of multienzyme complexes or for screening compounds for biological activity in vivo.

  Preferably, the animals of the invention are selected from mammals, preferably humans, rodents, pigs and cattle; C. elegans; and insects, preferably S. frugiperda and D. melanogaster.

  The tools of the present invention, ie polynucleotides, vectors, host cells and recombinant animals, allow convenient, rapid and simple modular binding of heterologous genes while minimizing the unique restriction sites required.

In another aspect, the present invention provides:
(A) a step of cloning one or more genes into MCS1 and / or MCS2 of the first vector of the present invention,
(B) Step of cloning one or more genes into MCS1 and / or MCS2 of the second vector of the present invention (c) Cutting out the entire functional arrangement at the restriction sites X and Y in the first transfer vector Process,
(D) cutting the second transfer vector with the growth module M,
(E) ligating the functional arrangement excised in step (c) to the propagation site of the second vector cleaved in step (d), thereby the first transfer vector propagation module; And (f) generating a third transfer vector having both expression cassettes of the second and second vectors, and (f) steps (a) to (e) for assembling transfer vectors having a plurality of expression cassettes. Optionally repeating
And a method for producing a multi-gene expression cassette.

  Preferably, the restriction sites X and Y in step (c) are PmeI and AvrII, or isoschizomers of these enzymes, and the restriction sites of the growth module of the second vector in step (d) are BstZ17I, SpeI, Selected from ClaI and NruI, or isoschizomers of these enzymes.

  Most preferably, the baculovirus transfer vector pFBDM can be used in step (a) and / or step (b).

  Furthermore, most preferably, the baculovirus transfer vector pUCDM can be used in step (a) and / or step (b).

  For further understanding of the general inventive method, an exemplary method using the preferred vector pFBDM is shown in FIG.

  In the method of the present invention, genes can be conveniently introduced into one or more expression cassettes of the vectors of the present invention, facilitating subsequent modular assembly of these expression cassettes into a single vector, while Minimizing the number of restriction sites required to do so.

A further aspect of the invention is:
(A) producing a multi-gene expression cassette of the present invention as defined above, and (b) introducing the multi-gene expression cassette into a host cell capable of simultaneously expressing the genes,
And a method for producing a multiprotein complex in vitro.

  Preferably, the introduction in step (b) is accomplished using a vector, preferably a virus.

  Preferably, the host cell is selected from mammals, yeast, E. coli and insect cells.

  When the host cell is an insect cell, S. frugiperda cells, more preferably Sf9, Sf21, Express Sf +, or High Five H5 cells, or D. melanogaster cells, more preferably S2 Schneider cells are preferred.

  When the host cell is a mammalian cell, HeLa, Huh7, HEK293, HepG2, KATO-III, IMR32, MT-2, pancreatic β cell, keratinocyte, bone marrow fibroblast, CHP212, primary neuron, W12, SK-N-MC, Saos-2, WI38, primary hepatocytes, FLC4, 143TK-, DLD-1, fetal lung fibroblasts, primary foreskin fibroblasts, Saos-2 osteosarcoma, MRC5, and MG63 cells Human cells selected from the group are preferred.

  When the host cell is a yeast cell, S. cerevisiae, S. pombe, C. albicans, or P. pastoris is preferred.

  The present invention is not limited to in vitro methods.

A different aspect of the present invention is:
(A) producing a multi-gene expression cassette according to claims 39 to 42, and (b) introducing the multi-gene expression cassette into an animal that simultaneously expresses the gene,
The present invention relates to a method for producing a multiprotein complex in vivo.

  Preferably, the introduction in step (b) is accomplished using a vector, preferably a viral vector, more preferably a baculovirus vector.

  As mentioned above, baculovirus is particularly useful as a gene delivery vehicle for mammalian cells.

  In order to produce a multiprotein complex in vivo, the animal is preferably selected from mammals, C. elegans, or insects.

  Preferred insects for carrying out the method are S. frugiperda and D. melanogaster.

  Preferred mammals for practicing the present invention are humans, rodents, pigs, or cows.

  In recent studies of multiprotein complexes (“interactomes”) that are increasing in eukaryotic cells, in particular, protein complexes that are intracellular quantities that are unsuitable for extraction from sources (eg, almost all “ There is a need for improved heterologous expression techniques for "human" multiprotein complexes). Eukaryotic proteins are often large (> 100 kDa) multidomain entities and are present in cells as a complex of an average of 5-8 proteins. In general, protein complexes containing large proteins cannot be produced in a functionally active state with conventional (eg, prokaryotic) expression techniques. Furthermore, the components of a multiprotein assembly often exhibit holding problems and impaired biological activity when it is isolated and manufactured. Current eukaryotic expression technologies (such as baculovirus systems, yeast and mammalian expression systems) are not designed to express multiple large genes encoding multiprotein complexes rapidly and flexibly.

  However, the vector of the present invention is prepared so as to satisfy these requirements. This vector can carry multiple gene expression cassettes for insertion into, for example, modified baculoviruses with improved protein production properties through independent entry sites (Cre-loxP and Tn7 and / or others). . Coupled with the high level of production rates obtained, for example, in insect cells, the vectors of the present invention can therefore be used for structural and functional studies providing important information for use in research and drug development, for example multiprotein complexes. As a physiologically useful target for discovering ligands (inhibitors, antibodies, etc.) that affect body activity, it can be used in a flexible manner to produce multiprotein complexes.

  The present invention is also particularly useful for producing vaccines against multi-subunit protein complexes in mammals. Complex multiprotein units often present different epitopes compared to individual proteins, and thus closely resemble related epitopes in many naturally occurring protein complexes, and thus are associated with antibody production. This would provide a better antigen target. In addition, multivalent vaccines and optionally multiple proteins for further protein adjuvants can be introduced into a single vector and expressed simultaneously to ensure maximum efficiency.

  Recently, virion-like particles (VLPs) consisting of four proteins from severe acute respiratory syndrome (SARS) coronavirus have been produced using recombinant baculovirus expression vectors (Mortola et al. Efficient assembly and release of SARS coronavirsus− like particles by a heterologous expression system FEBS Lertters 576, 174-178 (2004)), a potential vaccine for this disease. By using the vectors of the present invention, the production of such vaccines consisting of much more components than SARS-VLP is greatly enhanced due to their modular ability to carry multiple gene expression units. It will be easy.

Thus, the present invention also provides
(A) administering at least one polynucleotide and / or vector of the present invention to a mammal, whereby at least one protein encoded by at least one expression cassette of said polynucleotide is expressed in said animal )
(B) optionally administering an adjuvant to the animal, and (c) optionally isolating an antibody specific for the at least one protein and / or spleen cells producing the antibody,
And a method for producing a vaccine.

  The present invention provides a simple and simple protocol for recombinantly producing multiprotein complexes found in nature. These multiprotein complexes can be analyzed for protein complex interactions or protein modifications. These multiprotein complexes can also be analyzed for interactions with compounds with potential biological activity or even therapeutic efficacy.

  In another aspect, the present invention is directed to a method for analyzing protein complex interactions or protein modifications.

In a preferred embodiment, the present invention provides:
(A) providing a host cell comprising at least one polynucleotide and / or vector of the invention, said host cell comprising at least one expression cassette carrying genes for at least two proteins of interest; And (b) detecting an interaction or modification in one or more expressed proteins; and
To a method for screening protein complex interactions or protein modifications in vitro.

In another preferred embodiment, the invention relates to whether (i) a protein complex interaction is possible, or (ii) a protein (s) (of a multiprotein complex) is capable of being modified, Or (iii) an in vitro screening method for candidate compounds capable of inhibiting protein complex interactions, or (iv) capable of inhibiting modification of protein (s),
(A) providing a host cell comprising at least one polynucleotide of the present invention and / or a vector of the present invention, said host cell comprising at least one expression cassette having genes for at least two proteins of interest; And can express the protein simultaneously),
(B) administering a candidate compound to the host cell of step (a), and (c) detecting a protein complex interaction or modification in one or more expressed proteins, or protein complex Detecting inhibition of body interaction or inhibition of protein modification,
Including the method.

  The invention also encompasses in vivo screening assays.

  The polynucleotides and / or vectors of the invention can also be used for in vivo screening of protein-protein interactions or multiprotein complex-multiprotein complex interactions (as a general illustration of such screening methods, See FIG. 13I), and further for in vivo screening from randomized libraries or cDNA pools, as well as physiological modifications (phosphorylation, glycosylation) of specific protein complexes or multiprotein assemblies. Can be used for in vivo screening (see FIG. 13II for a general illustration of such screening methods).

In a further preferred embodiment, the invention provides that (i) a protein complex interaction is possible, or (ii) a protein (s) (of a multiprotein complex) is capable of modification, or (Iii) in vivo screening methods for candidate compounds capable of inhibiting protein complex interactions, or (iv) capable of inhibiting modification of protein (s),
(A) preparing an animal comprising at least one polynucleotide of the present invention and / or a vector of the present invention, said animal comprising at least one expression cassette having genes for at least two proteins of interest And can express the protein simultaneously),
(B) administering a candidate compound to the animal of step (a), and (c) detecting a protein complex interaction or modification in one or more expressed proteins, or a protein complex Detecting inhibition of interaction or inhibition of protein modification;
Is taught.

  Biologically active multiprotein complexes, as well as medically advantageous combinations of proteins, such as antibody mixtures, optionally with interleukins and / or adjuvants, are the polynucleotides of the invention and / or the gene delivery of the invention A vector can be used to administer to a mammal simultaneously.

  Accordingly, in a further aspect, the present invention also relates to the use of the polynucleotides and / or vectors of the present invention for the preparation of a medicament comprising a multi-gene transfer vehicle for gene therapy.

  Great efforts have been made to develop gene delivery systems for therapeutic applications. Gene therapy has traditionally been the subject of criticism, but has also been a very strong interest and interest. To date, significant progress has been made in evaluating gene therapy in clinical trials aimed at achieving in vivo and ex vivo strategies for safe and applicable clinical treatment for human disease (Worgall S. A realistic chance for gene therapy in the future. Pediatr. Nephrol. (2004)). In general, gene therapy currently exists as a very promising tool for gene modification and acquired diseases that require sustained or transient expression of therapeutic gene products (Worgall S. supra). reference). Recombinant virus-based vectors, particularly those that are not replicable in mammalian hosts (eg, baculovirus vectors), have recently emerged as powerful tools for mammalian cellular gene delivery, and include humans, primates, rodents. Have been successfully applied to all types of mammalian cell lines, including cattle, pigs and sheep (see Kost and Condreay. Recombinant baculoviruses as mammalian cell gene-delivery vectors. Trends Biotechnol. 20, 173-180 (2002). thing). Polycistronic to achieve more powerful results with combined gene therapy than with single gene therapy, both ex vivo and in vivo, to achieve combined gene transfer / therapeutic effects There is an increasing demand for viral vectors (de Felipe, P. Polycistronic viral vectors. Curr. Gene Ther. 2, 355-378 (2002); Planelles, V. Hybrid lentivirus vectors. Methods Mol. Biol. 229, 273 −284 (2003)). Also, the need for incorporation of an auxiliary gene to be administered in vivo into a carrier virus, eg, to inhibit inactivation by the complement system, is also associated with the baculovirus gp64 envelope carrying the human degradation promoter protein domain fusion. It has been demonstrated by using pseudotyped baculovirus with protein (Hueser et al., Incorporation of decay-accelerating factor into the baculovirus envelope generates complement-resistant gene transfer vectors. Nat. Biotechnol. 19, 451-455 ( 2001))), the need to provide recombinant modifications at the level of virus production in addition to multiple genes introduced for therapeutic purposes.

  Preferably, the vector used to prepare the drug is a recombinant baculovirus.

More preferably, the vector used to prepare the medicament of the present invention is
(I) one or more gene products for treatment, preferably proteins, and (ii) one or more baculovirus proteins,
A baculovirus comprising at least one multi-gene expression cassette that encodes a therapeutic gene product, preferably a protein, under the control of the mammalian promoter (s) activity of the animal to be treated And a gene for a baculovirus protein can be expressed under the control of a baculovirus promoter.

  In a preferred embodiment, the protein (s) of (ii) is a humanized baculovirus protein expressed from pseudotyped baculovirus, preferably a humanized baculovirus envelope protein gp64, for example, Gp64 fused with human proteins such as degradation promoting factors.

  In a more general aspect, the present invention is directed to the use of the polynucleotides and / or vectors and / or host cells and / or animals of the present invention for producing multiprotein complexes by recombinant techniques.

  Using the genes encoding the fluorescent proteins EYFP and ECFP surprisingly shows a nearly linear increase in fluorescent protein production with the number of genes encoding this protein incorporated into the vectors of the present invention. It was. More precisely, the production yield of fluorescent protein (per 1 L of insect culture cells) is 6-8 mg / in when there is one copy of the gene present in the vector of the invention used for expression. L, about 11 mg when there were 2 copies, and about 18 mg when there were 3 copies. This suggests an unprecedented and simple way to dramatically increase the amount of recombinant protein expressed using the expression system of the present invention, i.e. only one Instead of a gene, only two, three, or more copies of the same gene need be provided. For example, for protein production on an industrial scale, such as membrane proteins (e.g. GPCR), which are of greatest concern in drug development, for formulation purposes, in particular their production is very hindered by low production yields, This is particularly interesting.

  Furthermore, the recombinant multiprotein complex produced by the present invention is based on biophysical analysis, structural analysis, crystallographic analysis, electron microscopy analysis, and NMR of the multiprotein complex for research and even drug development. Very useful for analysis.

  Preferably, the invention relates to the use of the polynucleotides and / or vectors and / or host cells and / or animals of the invention for producing a vaccine.

  More preferably, for protein-related, protein modification, or biological effects in the host cell or animal, or for inhibition of protein-related, protein modification, or biological effects in the host cell or animal. Therefore, the use of the polynucleotides and / or vectors and / or host cells and / or animals of the invention for screening at least one protein in vitro or in vivo, optionally with candidate compounds .

  In another aspect, the invention includes at least a polynucleotide and / or vector and / or host cell of the invention, optionally further comprising instructions, antibiotics, buffers, and / or marker compounds. Good, kit of parts.

  In the following, specific embodiments of the invention will be described by way of example, but these examples should not be construed as limiting the scope of the invention.

[Example]
1. Novel baculovirus tools for multigene applications The following describes in detail the novel baculovirus transfer vectors of the present invention, specifically constructed for multigene applications. These transfer vectors present modified recipient baculovirus DNA that has been modified to enhance protein production, and two access to this baculovirus DNA in E. coli cells prepared for this purpose. It presents a simple and quick way to integrate genes through sites (attTn7 and LoxP). The vectors (pFBDM and pUCDM) contain a growth module that greatly facilitates modular binding of heterologous genes while minimizing the required unique restriction sites. The viral promoters of these vectors (currently p10 and polh late promoters) can be exchanged for other promoter sequences if desired. Similarly, the termination sequence (currently SV40, HSVtk) can be replaced.

  These vectors contain a modified baculovirus genome (MultiBac) with improved protein production properties. Two baculovirus genes were disrupted, resulting in increased retention of intracellular compartments during infection and protein production. The amount of protein produced by this system has been significantly improved through a reduction in virus-dependent proteolytic activity and a reduction in cell lysis.

  The vectors of the present invention allow a novel method for the rapid production of recombinant baculovirus DNA by accessing the viral genome through two specific sites (see FIG. 1 for review). ). In addition to the commercially available Tn7 transposon site, the LoxP sequence was introduced into another site on the baculovirus genome that can accept multiple gene expression cassettes from the pUCDM plasmid by site-specific recombination. Protocols have been developed to fully perform Tn7 transfer (from pFBDM derivatives carrying multiple gene cassettes) and LoxP site-specific recombination (from pUCDM derivatives carrying multiple gene cassettes) in a single step in E. coli. These protocols not only incorporate multigene cassettes with coding sequences for multiprotein complex subunits into MultiBac, but also incorporate specific enzymes (kinases, acetylases, etc.) to modify the protein under study. Also useful.

[Example 1: Transfer vectors pFBDM and pUCDM, and baculovirus vector MultiBac]
The transfer vector pFBDM (SEQ ID NO: 1, FIG. 11) contains two expression cassettes with multiple cloning sites MCS1 and MCS2 flanked by polh or p10 promoter and SV40 or HSVtk polyA signal sequence, respectively, head-to-head. Includes in the arrangement. Growth module M is located between the promoter sequences. The sequences used for Tn7 transfer (Tn7L and Tn7R) include an expression cassette and a gentamicin resistance marker. See Figure 2 for more details.

  The transfer vector pUCDM (SEQ ID NO: 2, FIG. 12) has the same expression cassette containing the same growth module as pFBDM. This expression cassette is adjacent to the LoxP inverted repeat. The vector pUCDM contains a chloramphenicol resistance marker and an R6Kγ conditional origin of replication that causes pir gene expression dependent growth in prokaryotic hosts. See Figure 3 for more details.

  In summary, both vectors were made as follows. The transfer vector pFBDM was obtained from pFastBac® DUAL (Invitrogen). The sequence 5'-TACTAGTATCGATTCGCGACC was inserted into the BstZ17I site between the polh promoter and the p10 promoter to produce a growth module that in turn contains BstZ17I, SpeI, ClaI, and NruI cleavage sites. A site for PmeI, a rare cutter, was added by replacing the SnaBI recognition sequence with the oligonucleotide 5'-AGCTTTGTTTAAACAAAGCT. After treatment with Mung Bean Nuclease (New England Biolabs), the vector pUCDM is generated by ligating the 998 bp EcoRV / AlwNI fragment of the pUNI10 univector containing the R6Kγ base and the 1258 bp AlwNI fragment from pLysS (Novagen). did. This pLysS fragment contains a chloramphenicol resistance marker. The resulting construct was digested with SmaI and XbaI and ligated to the 1016 bp PmeI / AvrII fragment of pFBDM. Unique restriction sites for the AvrII and PmeI restriction endonucleases were reintroduced by site-directed mutagenesis using the Quick Change protocol (Stratagene). Vectors pUCDM and pFBDM contain the same expression cassette containing the growth module.

This baculovirus vector, so-called MultiBac, means a bacmid containing an origin of replication (F-replicon) that allows its growth in E. coli strains (eg, DH10α and its derivatives) and It was constructed. Plasmid pKIloxP (which contains linear fragments used to replace the bMON14272 v-cath and chiA genes by ET recombination) was constructed from pFastBac® DUAL by recircularization of the 3385 bp StuI / FspI fragment. , Thereby removing these sites and destroying the ampicillin resistance gene. This pKI vector has the original pFastBac® DUAL p10 MCS containing the gentamicin marker and XhoI, NcoI and KpnI restriction sites. To obtain pKIloxP, pKI was modified as follows. A 1008 bp BspHI fragment of pFastBac® DUAL containing an ampicillin resistance marker was introduced into the NcoI site. Next, the homologous region HomA of the baculovirus chiA gene was amplified from bMON14272 using the primers 5′-GCGCGCCTCGAGGCCTCCCACGTGCCCGACCCCGGCCCG and 5′-GCGCGCCTCGAGGGAGGAGCTGCGCGCAATGC and digested with XhoI. The resulting 408 bp fragment was inserted into the XhoI site of pKI. The homologous region HomB of the v-cath gene was amplified using primers 5′-GCGCGCGGTACCGCGTTCGAAGCCATCATTA and 5′-GCGCGCGGTACCAGGCCTGAAAAATCCGTCCTCTCC, digested with KpnI, and the resulting 372 bp fragment was inserted into the pKI KpnI site. Finally, the Cre-loxP sequence was introduced into the NheI site by oligonucleotide 5′-CTAGAATAACTTCGTATAGCATACATTATACGAAGTTAGTTTAAACT. A gene encoding a Cre recombinase and 6 histidine tags immediately adjacent to the start codon was made from a synthetic oligonucleotide (Microsynth, CH) and ligated into a pBAD22 vector digested with NcoI / KpnI. Ampicillin resistance markers were inactivated by FspI / ScaI digestion. A zeocin resistance gene under the control of the synthesized prokaryotic promoter (EM7) was excised from pPICZA (Invitrogen) by PvuII / EcoRV digestion and ligated to a 5388 bp FspI / ScaI fragment to produce pBADZ-His ₆ Cre.

Transfer vectors pFBDM, pKILoxP and pBADZ-His ₆ Cre are propagated in common E. Coli strains such as Top10 cells (Invitrogen), pUCDM is a bacterial host, BW23473 and BW23474 expressing the pir gene Grow with.

Next, to create the baculovirus bacmid MultiBac, the vector pBAD-ETγ carrying the truncated recE under the arabinose-inducible PBAD promoter and recT under the EM7 promoter was converted to FspI as described for pBADZ-His ₆ Cre. and modified by into ScaI site put zeocin resistance gene from pPICZA, yielded pBADZ-ET ^~ γ. This vector was transformed into DH10BAC® (Invitrogen) cells providing bacmid bMON14272 and helper plasmid pMON7124 to form colonies resistant to kanamycin, tetracycline and zeocin. Single colonies were used to inoculate 500 ml of 2 × TY (1.6% bactotryptone, 1% yeast extract, 0.5% NaCl) in the presence of these antibiotics at 20 ° C. At OD ₆₀₀ = 0.25, L-arabinose (Fluka BioChemicals) was added to 0.1% and the culture was incubated to OD ₆₀₀ = 0.5. Electrocompetent DH10BACET cells were then generated according to standard procedures and stored at −80 ° C. Vector pKIloxP was transformed into JM110 cells (Stratagene) lacking dam and dcm activity. Unmethylated plasmid DNA was digested with StuI to generate an 1827 bp fragment containing an ampicillin resistance marker and a Cre-loxP sequence (close to the chiA and v-cath homology regions). A minimum of 0.3 μg of linear DNA was then electroporated into DH10BACET cells. Transformed cells were grown at 37 ° C. for 4 hours and plated on agar plates containing kanamycin, tetracycline and ampicillin. PCR with 5'-AGGTACTAAATATGGCG and 5'-CTGAGCGACATCACT, primers that anneal bacmid DNA from two single, triple-resistant colony-resistant colonies PCR fragments were analyzed by amplification and by sequencing PCR fragments to confirm correct integration. Frozen electrocompetent DH10MultiBac cells containing a modified bacmid in which the v-cath and chiA genes were replaced with ampicillin markers and LoxP were prepared as described above in culture using zeocin instead of ampicillin.

In order to create a composite MultiBac bacmid, pBADZ-His ₆ Cre was transformed into DH10MultiBac by electroporation (to express Cre recombinase) to form kanamycin, tetracycline and zeocin resistant colonies. A single colony was used to inoculate 500 ml of 2 × TY at 20 ° C. in the presence of these antibiotics. L-arabinose was added to 0.1% at OD ₆₀₀ = 0.25. Frozen electrocompetent DH10MultiBac ^Cre cells containing OD ₆₀₀ = 0.5 and overexpressed Cre recombinase were prepared. Plasmid pUCDM or its derivative was electroporated into DH10MultiBac ^Cre cells and spread on agar containing kanamycin, tetracycline and chloramphenicol. When colonies resistant to the three antibiotics were analyzed for integration of the lacZα gene and Tn7 on plates containing IPTG and Bluogal, blue colonies formed in all cases tested. Colonies were analyzed for the accuracy of the Cre-loxP site-specific recombination event by PCR amplification and sequencing as described above. The loss of pBADZ-His ₆ Cre was confirmed by replating in the presence of zeocin. Electrocompetent cells were prepared from individual clones in the presence of kanamycin, tetracycline and chloramphenicol. Transfer of the Tn7 element from the pFBDM derivative to Tn7, identification of the recombinant by blue / white screening, and transfection of the purified complex bacmid were performed according to convention (eg O'Reilly, DR, Miller, LK & Luckow , VA "Baculovirus expression vectors. A laboratory manual." Oxford University Press, New York-Oxford (1994); "Bac-to-Bac ^™ Baculoviorus Expression Systems Manual." Invitrogen, Life Technologies Incorporated (2000)). At least 1 μg each was used for co-transformation of DH10MultiBac ^Cre with pUCDM and pFBDM derivatives.

  The function of the F-replicon on the bacmid is to limit the copy number to one or two, reducing the possibility of unwanted recombination. By introducing the pFBDM derivative into MultiBac, the lacZα gene is destroyed, and cells that contain only the complex bacmid can be clearly identified. However, in the case of Cre-catalyzed fusion of pUCDM derivatives, the coexistence of both copies of the complex and the parent bacmid cannot be ruled out based on chloramphenicol resistance. Therefore, the virus from the first transfection with MultiBac containing the EYFP gene inserted by Cre catalyst was colonized by plaque purification. Since 29 (91%) of the 32 plaque-purified samples expressed EYFP, Cre-loxP site-specific recombination was performed with a nearly saturated efficacy. In spite of the presence of multiple copies of the viral promoter (p10, polh) and terminator (SV40, HSVtk), the MultiBac derivative is continuously passaged at least 5 times in Sf21 cells with a multiplicity of infection (MOI) ≦ 1. I was able to do it.

[Example 2: Preparation of multiple gene expression cassette]
The vectors pFBDM and pUCDM are particularly suitable for making multigene expression cassettes due to the growth module inserted between two promoters. The growth logic is illustrated in FIG. The only prerequisite for assembling multiple gene expression cassettes is that the restriction enzymes used for propagation (eg, PmeI, AvrII, SpeI, and either BstZ17I or NruI) are unique, A compound capable of masking additional sites that should not be cleaved, eg, by performing site-directed mutagenesis prior to assembly of multiple gene cassettes (eg, peptide nucleic acids) Can be easily achieved. The gene is cloned into MCS1 and MCS2 of pFBDM. The entire expression cassette is then excised by PmeI and AvrII digestion. The resulting fragment is placed in the propagation module of pFBDM containing an additional set of genes via either SpeI / BstZ17I or SpeI / NruI sites. SpeI produces sticky ends that are compatible with AvrII, while BstZ17I, NruI and PmeI are blunt cutters. In the process, the restriction sites involved are removed and growth can be repeated by repeatedly using the modules present in the inserted cassette. Similar logic also applies to the generation of pUCDM derivatives with multiple gene expression cassettes. In addition, the promoter and termination sequence can be readily altered using appropriate restriction sites in the vectors of the invention, if desired.

[Example 3: Baculovirus modified to enhance protein production]
The baculovirus genome was modified to obtain improved protein production characteristics. Disrupting the two baculovirus genes, v-cath and chiA, resulted in increased retention of intracellular compartments during infection and protein production. The v-cath gene encodes the viral protease V-CATH, which activates cell death through a process dependent on chiA (encoding chitinase), a nearby gene on viral DNA. Both genes were disrupted so that chitin-affinity chromatography for purification could be used selectively to eliminate V-CATH activity and not interfere with the chiA gene product. The properties of the protein produced by this so-called MultiBac baculovirus are markedly improved through a decrease in virus-dependent proteolytic activity and a decrease in cell lysis. Instead of the disrupted viral DNA sequence, a LoxP sequence for cre-lox site-specific recombination was included. See FIG. 5 for more details.

[Example 4: Cloning into pFBDM or pUCDM transfer vector]
Selected genes are cloned into pFBDM and pUCDM multicloning sites MCS1 or MCS2 (see FIGS. 2 and 3) using standard cloning procedures. Ligation reactions for pFBDM derivatives are transformed into standard E. coli cells for cloning (eg, TOP10, DH5α, HB101) and plated on agar containing ampicillin (100 μg / ml). The ligation reaction for the pUCDM derivative is transformed into E. coli cells expressing the pir gene (eg, BW23473, BW23474) and plated on agar containing chloramphenicol (25 μg / ml). The correct clone is selected based on specific restriction digests and DNA sequencing of the insert.

[Example 5: Cre-lox site-specific recombination protocol]
Approximately 5-10 ng of pUCDM derivative is incubated on ice (15 minutes) with 50-100 μl of electrocompetent DH10MultiBac ^Cre cells. Following electroporation (200Ω, 25 μF, 1.8 kV pulse), cells were incubated for 8 hours at 37 ° C., kanamycin (50 μg / ml), chloramphenicol (25 μg / ml) and ampicillin (100 μg / ml). Sprinkle on agar containing. Colonies appear after incubation at 37 ° C. (12-15 hours). The bacmid can then be prepared for insect cell transfection (see III.6.) Or the colony can be prepared for incorporation of pFBDM derivatives by Tn7 transfer (see below).

[Example 6: Transfer protocol for pFBDM derivatives]
Approximately 5-10 ng of pFBDM derivative is incubated on ice (15 minutes) with 50-100 μl of electrocompetent DH10MultiBac ^Cre cells. Following electroporation (200Ω, 25 μF, 1.8 kV pulse), cells were incubated for 8 hours at 37 ° C., and kanamycin (50 μg / ml), gentamicin (7 μg / ml), ampicillin (100 μg / ml), tetracycline ( 10 g / ml), BluoGal (100 μg / ml) and IPTG (40 μg / ml) on agar. After incubation at 37 ° C. (18-24 hours), white colonies are selected. The bacmid can then be prepared for insect cell infection (see below).

[Example 7: One-step transfer / cre-lox site-specific recombination]
Cre-lox site-specific recombination and Tn7 transfer can be performed simultaneously in DH10MultiBac ^Cre cells, if desired. Since the efficiency of the two transformations is reduced compared to transformation with only one plasmid, a very large amount of DNA must be used for this reaction. Approximately 2-4 μg of selected pFBDM derivative and 2-4 μg of pUCDM derivative are incubated on ice (15 minutes) with 50-100 μl of electrocompetent DH10MultiBac ^Cre cells. Following electroporation (200Ω, 25 μF, 1.8 kV pulse), cells were incubated for 8 hours at 37 ° C., and chloramphenicol (25 μg / ml), kanamycin (50 μg / ml), gentamicin (7 μg / ml) , Spread on agar containing tetracycline (10 μg / ml), BluoGal (100 μg / ml) and IPTG (40 μg / ml). After incubation at 37 ° C. (18-24 hours), white colonies are selected. The bacmid can then be prepared for insect cell infection (see below).

Example 8: Preparation of cre-lox incorporation for Tn7 transition
For certain applications, it may be advantageous to add protein genes for expression to the DNA of MultiBac baculovirus already carrying a set of foreign genes incorporated by Cre catalyst. DH10MultiBac ^Cre cells (see III.2.) That provide recombinant MultiBac with an incorporated pUCDM derivative were then chloramphenicol (25 μg / ml), kanamycin (50 μg / ml), ampicillin (100 μg / ml). ml), tetracycline (10 μg / ml), BluoGal (100 μg / ml) and IPTG (40 μg / ml) on the agar again. The blue colonies with metastatic-competent MultiBac derivatives are then collected until OD ₆₀₀ reaches 0.5, chloramphenicol (25 μg / ml), kanamycin (50 μg / ml), ampicillin (50 μg / ml) and tetracycline. Incubate at 37 ° C. in 500 ml of 2 × TY medium containing (10 μg / ml). The culture is then cooled on ice (15 minutes) and centrifuged (4000 rpm, 8 minutes). The cell pellet is resuspended in 250 ml of cold 10% glycerol solution (sterile) and centrifuged (4000 rpm, 8 minutes). The cell pellet is then resuspended in 200 ml of chilled 10% glycerol solution (sterile) and centrifuged again (4000 rpm, 8 minutes). The cell pellet is then resuspended in 50 ml of chilled 10% glycerol solution (sterile), centrifuged, and finally resuspended in 1 ml of 10% glycerol solution (sterile). The cells are frozen in aliquots of 50-100 μl in liquid nitrogen and stored at −80 ° C. The transfer of the pFBDM derivative can be performed as follows.

[Example 9: Preparation of bacmid and infection of insect cells]
Bacmid DNA preparation, insect cell infection, and protein expression are performed according to conventional practices (eg, O'Reilly, DR, Miller, LK & Luckow, VA "Baculovirus expression vectors. A laboratory manual." Oxford University Press, New York-Oxford (1994); "Bac-to-Bac ^™ Baculoviorus Expression Systems Manual." Invitrogen, Life Technologies Incorporated, (2000)).

[Example 10: Efficiency of cre-lox site-specific recombination into MultiBac]
MultiBac growth in DH10MultiBac ^Cre cells is determined by the presence of the F-replicon on the bacmid. The function of the F-replicon is strict control of copy number (one or two), reducing the possibility of unwanted recombination. Introduction of the pFBDM derivative into MultiBac disrupts the lacZα gene, thus allowing clear identification of cells containing only complex bacmids. However, in the case of incorporation of pUCDM derivatives by Cre catalyst, the coexistence of one complex bacmid and one parent MultiBac molecule cannot be ruled out based on chloramphenicol resistance. Therefore, the virus from the first transfection with MultiBac containing the gene for the yellow fluorescent protein EYFP inserted by Cre catalyst was colonized by plaque purification. Since 29 (91%) of the 32 plaque-purified samples expressed EYFP, it can be said that the cre-lox site-specific recombination reaction occurred with almost saturated efficiency (see FIG. 6 for further details). )

[Example 11: Effect of chiA and v-cath deficiency in MultiBac]
During heterologous protein production using a commercial baculovirus expression system, a virus-dependent proteolytic breakdown consistent with the action of cysteine protease was observed. The baculovirus v-cath gene encodes a cysteine protease that is directly involved in the liquefaction of the insect cell host. V-CATH activates cell death through a process dependent on chiA (encoding chitinase), a contiguous gene on viral DNA. Disrupting both genes eliminates V-CATH activity and allows selective use of chitin affinity chromatography for purification that does not interfere with the chiA gene product. The effect of disruption was analyzed by comparing samples from cells infected with MultiBac virus with samples from cells infected with commercial baculovirus carrying v-cath and chiA. In the lysates of cells infected with MultiBac, a significant decrease in proteolytic activity was observed while the maintenance of cell integrity was greatly enhanced (more specifically, (See FIG. 7).

[Example 12: Production of 275 kDa protein complex using MultiBac]
In a test experiment, a yeast Isw2 chromatin reconstitution complex consisting of a 130 kDa Isw2p protein and a 145 kDa Itc1p protein was expressed. The first MultiBac was constructed by incorporating both proteins into the attTn7 site, and the second MultiBac was constructed by incorporating Isw2p into the attTn7 site and inserting Itc1p into the LoxP site. Spatial disruption of two genes on the virus does not adversely affect the relative production levels of subunits or functional assembly of the Isw2 complex in insect cells, and Cre for cointegration and protein expression -Supports the usefulness of both lox and Tn7 sites. See FIG. 8 for more details.

[Example 13: Production of multiprotein complex using MultiBac]
In addition to the yeast Isw2 reconstitution complex, a three member yeast Isw1b complex (360 kDa) and a human transcription factor complex (700 kDa) were expressed using MultiBac. Furthermore, in all cases, EYFP fluorescent protein was also co-expressed. In co-expression experiments, there was no change in the yield of EYFP and the yield of each complex, revealing that expression was not saturated in this system, and a larger complex containing more subunits. It is suggested that can be expressed using Multibac. See FIG. 9 for more details.

[Example 14: MultiBac for gene transfer in mammalian cells ("BacMam")
MultiBac derivative viruses containing fluorescent proteins EYFP and ECFP under baculovirus promoter control and dsRED under CMV late promoter control were prepared. Insect cells infected with this hybrid virus showed expression of EYFP and ECFP. Mammalian COS cells infected with amplified virus in insect cells showed strong fluorescence of the dsRED protein, demonstrating the availability of MultiBac (“BacMam”) for multigene transfer into mammalian cells. See FIG. 10 for more details.

II. MultiBac kits The MultiBac kits of the present invention may include one or more of the following:
DH10MultiBac ^Cre cells, BW23474, BW23474 cells ^+, pFBDM vector, pUCDM vector, control plasmid for Tn7 metastasis ^*, control plasmid ^*, typical transfection reagents for cre-lox site-specific recombination ^#
+; E. coli strain expressing the pir gene for growth of pUCDM derivative (any other strain with pir + background can also be used).
* In the experiment, vectors carrying genes for the fluorescent proteins ECFP and EYFP were used as controls. These genes are sold with permission by Molecular Probes. These are particularly useful as controls, since fluorescence observations either with a fluorescence microscope or with a fluorescence spectrophotometer are exclusively easy. However, any type of control plasmid carrying a gene encoding a readily identifiable protein (glucuronidase, catechol dioxygenase, XylE, luciferase, etc.) can also be used.
#: For example, CellFECTIN (Invitrogen), LipoTAXI (registered trademark) transfection reagent (Stratagene), etc.

Schematic diagram of the MultiBac system: The gene of interest is assembled into a multigene expression cassette using the growth module present on the transfer vectors pFBDM and pUCDM. The resulting vector is introduced into MultiBac baculovirus DNA in DH10MultiBac ^Cre E. coli cells containing factors for Tn7 transfer (for pFBDM derivatives) and cre-lox site-specific recombination (for pUCDM derivatives) . Colonies containing a bacmid carrying an integrated multigene cassette are identified by blue / white screening (Tn7 transfer disrupts the lacZα gene) and chloramphenicol resistance (given by Cre-catalyzed incorporation of pUCDM derivatives). Transfer and site-specific recombination can be performed sequentially or alternatively in a one-step reaction. Bacmid DNA is prepared from selected colonies and used for transfection into insect cells for protein production. Transfer vector pFBDM: Circular map of pFBDM shows promoter (polh, p10), terminator (SV40, HSVtk), multiple cloning sites (MCS1, MCS2), transposon elements (Tn7L, Tn7R) and resistance markers (ampicillin and gentamicin) ing. The gene of interest is cloned into MCS1 or MCS2 using a unique restriction site. The multiplication module (M) is located between the p10 promoter and the polh promoter. Transfer vector pUCDM: Circular map of pUCDM consists of promoter (polh, p10), terminator (SV40, HSVtk), multiple cloning site (MCS1, MCS2), inverted repeat (LoxP) for cre-lox site-specific recombination and The resistance marker (chloramphenicol) is shown. The gene of interest is cloned into MCS1 or MCS2 using a unique restriction site. The growth module (M) is located between the p10 promoter and the polh promoter. Assembly of multiple gene expression cassettes: Growth logic is shown for pFBDM. An expression cassette containing the two selected genes (indicated by a and b) is excised by digestion with AvrII and PmeI (enclosed), and BstZ17I / SpeI (or alternatively present in the proliferation module (M)) (or Alternatively, the construct containing the additional gene (c, d) is inserted into the growth module via the NruI / SpeI) site (Ligation). SpeI produces sticky ends that are compatible with AvrII, while BstZ17I, NruI and PmeI are blunt cutters. Multiple gene derivatives of pUCDM can be made according to similar logic. MultiBac baculovirus DNA: The modified viral genome is shown schematically. The Tn7 adhesion site is located within the LacZα gene; therefore, when sown on agar containing BluoGal and IPTG, insertion of the Tn7 element from the pFBDM derivative produces a white phenotype. In place of the disrupted v-cath and chiA virus genes, insertion of a LoxP sequence to allow the pUCDM derivative to be accepted by Cre catalyst produces a chloramphenicol resistant phenotype. Efficiency of cre-lox site-specific recombination in MultiBac: The gene encoding EYFP, a yellow fluorescent protein, was cloned into pUCDM and incorporated into MultiBac by Cre catalyst. Virus was colonically isolated by plaque assay. Thirty-two plaques were tested for EYFP expression by fluorescence spectroscopy. Excitation was 488 nm. Shown are spectra recorded from cell lysates of 1-12 viruses (top). In this experiment, 91% (29 out of 32) of the samples tested showed strong fluorescence emission indicating the expression of EYFP protein (lower table). Effect of v-cath and chiA on Multibac: The lysate of cells infected with MultiBac virus (BacLoxP) was compared with the lysate of cells infected with commercial baculovirus ("wild type") (Wildtype). In both samples, virus-dependent protein production is substantially the same 48 hours post infection (Figure A). It has been shown that incubation of cell lysates at room temperature for 96 hours after harvesting does not substantially change the protein content in lysate samples of cells infected with MultiBac. In contrast, proteolysis is evident in samples of cells infected with wild-type virus (Figure B). The effect of protease deficiency on cell morphology 72 hours after infection, micrographs of cells infected with MultiBac lacking v-cath and chiA (MultiBac; BacLoxP), or commercially available viruses containing protease and chitinase genes It shows by the microscope picture of the cell infected with (Wildtype). The cells in the upper micrograph appear to be a uniform circle and remain intact. In contrast, cell lysis is widespread in the lower micrograph. Expression of the 275 kDa chromatin remodeling complex Isw2 using MultiBac: The Isw2 complex was expressed from the attTn7 site or alternatively from the attTn7 and LoxP sites of MultiBac. Protein conjugates purified from both complex bacmids as well as cell lysates exhibit substantially identical protein production levels. Samples from uninfected Sf21 cells were included as a control. Expression of yeast Isw2, Isw1b and human TFIID subcomplex using MultiBac: The site of integration is shown schematically at the top of the Coomassie-stained gel section showing the components of each multiprotein complex. Yield is shown. The expression of EYFP is shown below by the yield calculated from the absorbance spectrum and the Western blot section of each cell lysate. Use of MultiBac for gene transfer in mammalian cells ("BacMam"): Hybrid virus (left side) results in EYFP and ECFP expression in insect cells (upper right side) and is infected with virus amplified in Sf21 cells Induced COS cells result in dsRED expression (bottom right). Circular baculovirus transfer vector pFBDM: FIG. 11 shows the complete polynucleotide sequence of the circular baculovirus transfer vector pFBDM according to a preferred embodiment of the present invention. Circular baculovirus transfer vector pUCDM: FIG. 12 shows the complete polynucleotide sequence of the circular baculovirus transfer vector pUCDM according to a preferred embodiment of the present invention. In Vivo Screening for Protein-Protein Interactions and Modifications Using MultiBac: FIG. 13 to evaluate physical binding of protein / protein complexes (I.) and / or their modifications (II.) In vivo Figure 2 illustrates a screening strategy using the vectors of the present invention.

Claims (25)

Formula (I) below

[This arrangement includes:
(A) at least two expression cassettes in head-to-head, head-to-tail, or tail-to-tail arrangement, T1-MCS1-P1 and P2-MCS2-T2; MCS1 or MCS2, which are multiple cloning sites, respectively MCS1 is adjacent to promoter P1 and termination sequence T1, and MCS2 is adjacent to promoter P2 and termination sequence T2.
(B) at least one growth module M (including at least two restriction sites A and B) between promoters P1 and P2,
(C) at least two restriction sites X and Y (each adjacent to one of the expression cassettes),
{Where:
(I) Restriction sites A and X and B and Y are compatible, but
(Ii) the ligation products of AY and BX are not enzymatically cleaved by a, b, x, or y, which are specific restriction enzymes for restriction sites A, B, X and Y, and (iii) restriction sites A and B and X and Y are not compatible}]
A polynucleotide for multiple gene applications, comprising a functional arrangement of   The restriction sites A and B of the growth module M are selected from the group consisting of BstZ17I, SpeI, ClaI and NruI, or a restriction enzyme (“isoschizomer”) having the same cleavage site as the enzyme. Polynucleotide.   The polynucleotide according to claim 1 or 2, wherein the restriction sites X and Y are selected from the group consisting of PmeI and AvrII, or a restriction enzyme ("isoschizomer") having the same restriction site as the enzyme. The promoters P1 and P2 are polh, p10 and p _XIV late baculovirus promoter, vp39 baculovirus late promoter, vp39polh baculovirus late / late hybrid promoter P _{cap / polh} , pcna, etl, p35, egt, da26 baculovirus early promoter; CMV, SV40, UbC, EF -1α, RSVLTR, MT, P DS47, Ac5, P GAL and is selected from the group consisting of P _ADH, claims 1 to 3 as described in any one of the polynucleotide.   The polynucleotide according to any one of claims 1 to 4, wherein the termination sequences T1 and T2 are selected from SV40, HSVtk, or BGH (bovine growth hormone).   The polynucleotide of any one of claims 1 to 5, further comprising at least one site for integration into a vector or host cell.   The integration site is a transposon element of Tn7, a λ-integrase specific adhesion site and SSR (site specific recombinase), preferably a cre-lox specific recombination (LoxP) site or a FLP recombinase specific recombination (FRT) site; The polynucleotide of claim 6 selected from the group consisting of: (A) promoters P1 and P2 selected from polh and p10,
(B) a termination sequence selected from SV40 and HSVtk,
(C) restriction sites A and B in the growth module M, selected from the group consisting of BstZ17I, SpeI, ClaI and NruI,
(D) restriction sites X and Y selected from the group consisting of PmeI and AvrII,
(E) a site for viral integration selected from cre-lox and Tn7 recombination systems;
The polynucleotide according to any one of claims 1 to 7, comprising:   A vector comprising the polynucleotide sequence of any one of claims 1-8.   Said viruses are adenovirus, adeno-associated virus (AAV), autonomic parvovirus, herpes simplex virus (HSV), retrovirus, radinovirus, Epstein Barr virus, lentivirus, Semliki Forest virus and baculovirus The vector according to claim 9, which is selected from the group consisting of:   The vector according to claim 10, wherein the vector is a baculovirus expression vector.   The vector according to claim 11, wherein the two baculovirus genes v-cath and chiA are functionally disrupted.   The vector according to any one of claims 9 to 12, further comprising a site for SSR (site-specific recombinase), preferably a LoxP site for cre-lox site-specific recombination.   14. A vector according to claim 12 or 13, wherein the cre-lox site is located in one or both of two baculovirus genes v-cath and chiA.   15. Vector according to any one of claims 9 to 14, comprising a transposon element, preferably a Tn7 adhesion site.   The vector of claim 9, wherein the vector has the sequence of SEQ ID NO: 1 (pFBDM).   10. The vector of claim 9, wherein the vector has the sequence of SEQ ID NO: 2 (pUCDM).   A host cell comprising the polynucleotide sequence according to any one of claims 1 to 8 and / or the vector according to any one of claims 9 to 17.   Mammalian cells, preferably human cells, rodent cells, pig cells, cow cells and sheep cells; C. elegans cells; yeast cells, preferably S. cervisiae, S. pombe, C. albicans and P. 19. A host cell according to claim 18 selected from the group consisting of pastoris; insect cells; and E. coli cells.   A recombinant animal comprising the polynucleotide sequence according to any one of claims 1 to 8 and / or the vector according to any one of claims 9 to 17.   21. An animal according to claim 20, selected from mammals, preferably humans, rodents, pigs and cows, and C. elegans. (A) a step of cloning one or more genes into MCS1 and / or MCS2 of the first vector according to any one of claims 9 to 17,
(B) a step of cloning one or more genes into MCS1 and / or MCS2 of the second vector according to any one of claims 9 to 17 (c) a restriction site X in said first transfer vector And Y to cut out the entire functional arrangement,
(D) cleaving the second transfer vector with the growth module M;
(E) ligating the functional arrangement excised in step (c) to the propagation site of the second vector cleaved in step (d), thereby the propagation module of the first transfer vector; Producing a third transfer vector having both expression cassettes of the first and second vectors; and (f) steps (a) to (a) for assembling transfer vectors having a plurality of expression cassettes. Optionally repeating (e),
A method for producing a multi-gene expression cassette. (A) producing a multi-gene expression cassette according to claim 22, and (b) introducing the multi-gene expression cassette into a host cell capable of simultaneously expressing the genes,
A method for producing a multiprotein complex in vitro, comprising: (A) producing a multi-gene expression cassette according to claim 22, and (b) introducing the multi-gene expression cassette into an animal that simultaneously expresses the gene,
A method for producing a multiprotein complex in vivo, comprising:   Use of the polynucleotide according to any one of claims 1 to 8 and / or the vector according to any one of claims 9 to 17 for the preparation of a medicament comprising a multi-gene transfer vehicle for gene therapy. .

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